The long range goal of this proposal is to identify the molecular bases for certain human congenital hemolytic anemias and to develop a detailed concept of the membrane skeleton's role in maintenance of red cell stability and shape. Two major immediate aims will be to identify precise mutations responsible for red cell membrane instability in certain hemolytic anemias and to elucidate mechanisms contributing to this destabilization using established and novel approaches. General clinical classifications of hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and hereditary pyropoikilocytosis (HPP) actually correspond to a large number of unrelated biochemical disorders. These disorders are poorly characterized biochemically and very little is known about the mechanisms involved in membrane destabilization. Spectrin, a complex, major protein with a central role in the red cell membrane skeleton, will be studied first and in the greatest detail. Red cell spectrin has recently been directly implicated in several unrelated hemolytic disorders and these initial observations will be extended. Effects of specific mutations on structural and functional properties, and their contributions to membrane destabilization will be investigated. Synthetic peptides and mutant peptides will be used in known functions. In conjunction with functional characterizations, mechanisms responsible for long range transducing effects in the spectrin molecule will be investigated. Current evidence indicates that mutations can be located substantial distances from the destabilized functional site. The basis for this remarkable long range effect is not known, although it is probably directly related to spectrin conformation. Further analysis of the 106 amino acid repeat unit and spectrin conformation will use new computer prediction methods coupled with synthetic peptide experiments to develop and test new models. Characterization of the precise mutations in selected hemolytic anemias coupled with more detailed analyses of structural and functional properties of proteins in the membrane skeleton will make vital contributions to a refined understanding of the role of the membrane skeleton in normal and diseased human red cells.